-Recent evidence indicates that oxidative stress is central to the pathogenesis of a wide variety of degenerative diseases, aging, and cancer. Oxidative stress occurs when the delicate balance between production and detoxification of reactive oxygen species is disturbed. Mammalian cells respond to this condition in several ways, among which is a change in mitochondrial morphology. In the present study, we have used rotenone, an inhibitor of complex I of the respiratory chain, which is thought to increase mitochondrial O 2 Ϫ ⅐ production, and mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to investigate the relationship between mitochondrial O 2 Ϫ ⅐ production and morphology in human skin fibroblasts. Video-rate confocal microscopy of cells pulse loaded with the mitochondria-specific cation rhodamine 123, followed by automated analysis of mitochondrial morphology, revealed that chronic rotenone treatment (100 nM, 72 h) significantly increased mitochondrial length and branching without changing the number of mitochondria per cell. In addition, this treatment caused a twofold increase in lipid peroxidation as determined with C11-BODIPY 581/591 . Finally, digital imaging microscopy of cells loaded with hydroethidine, which is oxidized by O 2 Ϫ ⅐ to yield fluorescent ethidium, revealed that chronic rotenone treatment caused a twofold increase in the rate of O 2 Ϫ ⅐ production. MitoQ (10 nM, 72 h) did not interfere with rotenone-induced ethidium formation but abolished rotenone-induced outgrowth and lipid peroxidation. These findings show that increased mitochondrial O 2 Ϫ ⅐ production as a consequence of, for instance, complex I inhibition leads to mitochondrial outgrowth and that MitoQ acts downstream of this O 2 Ϫ ⅐ to prevent alterations in mitochondrial morphology. rhodamine 123; video-rate confocal microscopy; superoxide; MitoQ HIGHLY AEROBIC CELLS from brain, heart, muscle, liver, kidney, and endocrine tissue depend on the ATP-generating capacity of their mitochondria to meet energetic demands. Acute changes in cellular energy consumption are met through feedback and/or feedforward regulation of enzymes involved in aerobic ATP production, whereas chronic changes lead to alterations in mitochondrial capacity and/or architecture (3,22,35,36).Marked changes in the structure of the cellular mitochondrial network are observed during differentiation, cellular senescence, and apoptosis, whereas subtle rearrangements occur during cellular growth and division (56).Mitochondria generate ATP through oxidative phosphorylation (OXPHOS), and defects in this system lead to decreased energy production, increased formation of O 2 Ϫ ⅐ and derived reactive oxygen species such as hydrogen peroxide and ⅐OH, and the release of death-promoting factors (44,47,56). Defects occur in a wide variety of degenerative diseases, aging, and cancer and primarily affect tissues that have high energy requirements and are unable to adapt to conditions of reduced mitochondrial energy supply. Cells that can survive under such conditions, ...
Complex I (NADH:ubiquinone oxidoreductase) is the largest multisubunit assembly of the oxidative phosphorylation system, and its malfunction is associated with a wide variety of clinical syndromes ranging from highly progressive, often early lethal, encephalopathies to neurodegenerative disorders in adult life. The changes in mitochondrial structure and function that are at the basis of the clinical symptoms are poorly understood. Video-rate confocal microscopy of cells pulse-loaded with mitochondria-specific rhodamine 123 followed by automated analysis of form factor (combined measure of length and degree of branching), aspect ratio (measure of length), and number of revealed marked differences between primary cultures of skin fibroblasts from 13 patients with an isolated complex I deficiency. These differences were independent of the affected subunit, but plotting of the activity of complex I, normalized to that of complex IV, against the ratio of either form factor or aspect ratio to number revealed a linear relationship. Relatively small reductions in activity appeared to be associated with an increase in form factor and never with a decrease in number, whereas relatively large reductions occurred in association with a decrease in form factor and/or an increase in number. These results demonstrate that complex I activity and mitochondrial structure are tightly coupled in human isolated complex I deficiency. To further prove the relationship between aberrations in mitochondrial morphology and pathological condition, fibroblasts from two patients with a different mutation but a highly fragmented mitochondrial phenotype were fused. Full restoration of the mitochondrial network demonstrated that this change in mitochondrial morphology was indeed associated with human complex I deficiency.
Background: Understanding the interdependence of mitochondrial and cellular functioning in health and disease requires detailed knowledge about the coupling between mitochondrial structure, motility, and function. Currently, no rapid approach is available for simultaneous quantification of these parameters in single living cells. Methods: Human skin fibroblasts were pulse-loaded with the mitochondria-selective fluorescent cation rhodamine 123. Next, mitochondria were visualized using video-rate (30 Hz) confocal microscopy and real-time image averaging. To highlight the mitochondria, the acquired images were binarized using a novel image processing strategy. Results: Our approach enabled rapid and simultaneous quantification of mitochondrial morphology, mass, potential, and motility. It was found that acute inhibition of
Coxsackievirus infection leads to a rapid reduction of the filling state of the endoplasmic reticulum (ER) and Golgi Ca 2؉ stores. The coxsackievirus 2B protein, a small membrane protein that localizes to the Golgi and to a lesser extent to the ER, has been proposed to play an important role in this effect by forming membrane-integral pores, thereby increasing the efflux of Ca 2؉ from the stores. Here, evidence is presented that supports this idea and that excludes the possibility that 2B reduces the uptake of Ca 2؉ into the stores. Measurement of intra-organelle-free Ca 2؉ in permeabilized cells revealed that the ability of 2B to reduce the Ca 2؉ filling state of the stores was preserved at steady ATP. Biochemical analysis in a cellfree system further showed that 2B had no adverse effect on the activity of the sarco/endoplasmic reticulum calcium ATPase, the Ca 2؉ -ATPase that transports Ca 2؉ from the cytosol into the stores. To investigate whether 2B specifically affects Ca 2؉ homeostasis or other ion gradients, we measured the lumenal Golgi pH. Expression of 2B resulted in an increased Golgi pH, indicative for the efflux of H ؉ from the Golgi lumen. Together, these data support a model that 2B increases the efflux of ions from the ER and Golgi by forming membrane-integral pores. We have demonstrated that a major consequence of this activity is the inhibition of protein trafficking through the Golgi complex.Enteroviruses (e.g. poliovirus, coxsackievirus, ECHOvirus) belong to the family of Picornaviridea, a large family of nonenveloped, cytolytic viruses that contain a single-stranded RNA genome of positive polarity. Upon infection, enteroviruses induce a number of dramatic alterations in their host cell, which serve to create the appropriate conditions for viral RNA replication and/or prevent antiviral host cell responses. One of these alterations is the modification of intracellular Ca 2ϩ homeostasis. We have previously shown that infection of HeLa cells with coxsackievirus results in a reduction of the amount of Ca 2ϩ that can be released from the intracellular stores using thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA), 2 the Ca 2ϩ -ATPase that transports Ca 2ϩ from the cytosol into the stores. In addition, a gradual increase in the cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) was observed due to the influx of extracellular Ca 2ϩ (1). The enterovirus 2B protein, one of the nonstructural proteins involved in viral RNA replication, plays a major role in the alterations in intracellular Ca 2ϩ homeostasis that take place in enterovirus-infected cells (1, 2). The mechanism by which 2B, or its precursor 2BC, exerts its effects is largely unknown. Ca 2ϩ homeostasis in the intracellular stores (i.e. endoplasmic reticulum (ER) and Golgi) is the net result of the activity of the SERCA on the one hand and the continuous passive Ca 2ϩ leak from these organelles that exists under normal conditions on the other hand (3). Thus, the reductions in the Ca 2ϩ filling state of the stores ...
Human mitochondrial complex I (NADH:ubiquinone oxidoreductase) of the oxidative phosphorylation system is a multiprotein assembly comprising both nuclear and mitochondrially encoded subunits. Deficiency of this complex is associated with numerous clinical syndromes ranging from highly progressive, often early lethal encephalopathies, of which Leigh disease is the most frequent, to neurodegenerative disorders in adult life, including Leber's hereditary optic neuropathy and Parkinson disease. We show here that the cytosolic Ca 2؉ signal in response to hormonal stimulation with bradykinin was impaired in skin fibroblasts from children between the ages of 0 and 5 years with an isolated complex I deficiency caused by mutations in nuclear encoded structural subunits of the complex. Inhibition of mitochondrial Na ؉ -Ca 2؉ exchange by the benzothiazepine CGP37157 completely restored the aberrant cytosolic Ca 2؉ signal. This effect of the inhibitor was paralleled by complete restoration of the bradykinin-induced increases in mitochondrial Ca 2؉ concentration and ensuing ATP production. Thus, impaired mitochondrial Ca 2؉ accumulation during agonist stimulation is a major consequence of human complex I deficiency, a finding that may provide the basis for the development of new therapeutic approaches to this disorder.Human mitochondrial complex I (NADH:ubiquinone oxidoreductase) is the largest multisubunit assembly of the oxidative phosphorylation (OXPHOS) 1 system, comprising 39 nuclear encoded and seven mitochondrially encoded subunits. Malfunction of this complex is associated with a wide variety of clinical syndromes ranging from often early lethal disorders, of which Leigh disease, a progressive encephalopathy, is the most frequent, to neurodegenerative disorders in adulthood, including Leber's hereditary optic neuropathy and Parkinson disease. In recent years, all human nuclear structural complex I genes have been characterized, which allowed us to elucidate the genetic defect in 40% of a cohort of complex I-deficient patients in which the enzyme defect was present in at least skeletal muscle and cultured skin fibroblasts (1-7). To enhance our understanding of the pathophysiological consequences of these diseases, with the final aim of developing new treatment strategies to stabilize or even cure these conditions, we study genetically characterized human complex I-deficient fibroblast cell lines as a model for OXPHOS system disease, knowing that these cells are glycolytic (8). Several hypotheses concerning the pathophysiology of OXPHOS diseases have been investigated of which the most consistent are (a) increased production of reactive oxygen species (9), (b) decreased potential across the mitochondrial inner membrane (10), (c) decreased intracellular ATP levels (11-13), and (d) altered Ca 2ϩ homeostasis (10, 12). In agreement with the reactive oxygen species hypothesis (a), we found that metallothioneins were up-regulated in all of the genetically characterized complex I-deficient cell lines (14). Pilot experiments w...
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